Photoimmunotherapy Using Cationic and Anionic Photosensitizer-Antibody Conjugates against HIV Env-Expressing Cells

Abstract

Different therapeutic strategies have been investigated to target and eliminate HIV-1-infected cells by using armed antibodies specific to viral proteins, with varying degrees of success. Herein, we propose a new strategy by combining photodynamic therapy (PDT) with HIV Env-targeted immunotherapy, and refer to it as HIV photoimmunotherapy (PIT). A human anti-gp41 antibody (7B2) was conjugated to two photosensitizers (PSs) with different charges through different linking strategies; "Click" conjugation by using an azide-bearing porphyrin attached via a disulfide bridge linker with a drug-to-antibody ratio (DAR) of exactly 4, and "Lysine" conjugation by using phthalocyanine IRDye 700DX dye with average DARs of 2.1, 3.0 and 4.4. These photo-immunoconjugates (PICs) were compared via biochemical and immunological characterizations regarding the dosimetry, solubility, and cell targeting. Photo-induced cytotoxicity of the PICs were compared using assays for apoptosis, reactive oxygen species (ROS), photo-cytotoxicity, and confocal microscopy. Targeted phototoxicity seems to be primarily dependent on the binding of PS-antibody to the HIV antigen on the cell membrane, whilst being independent of the PS type. This is the first report of the application of PIT for HIV immunotherapy by killing HIV Env-expressing cells.

Keywords: HIV immunotherapy; HIV-infected cell; monoclonal antibody; photodynamic therapy; photoimmunotherapy; phthalocyanine; porphyrin.

Figure 1

Structure and function of photo-immunoconjugates (PICs). (A) Anti-gp41 monoclonal antibody (7B2 MAb) conjugated with 4 porphyrins can specifically bind to the HIV infected cells expressing HIV Env (gp41 and gp120). The presence of CD4 may increase the binding ability of 7B2-porphyrin to the gp41 on the cell membrane. Porphyrins can be activated by visible light of a specific wavelength (herein 405 nm) to initially generate singlet oxygen or ROS reaction, and consequently kill the target cell. Furthermore, the emission of red fluorescent by porphyrin can be observed during irradiation. (B) Schematic picture demonstrates two generations of PICs in the study; Lysine modification leading to heterogeneous IR700-PICs with “average distribution” of DARs 2, 3 and 4 with undefined physical and pharmacokinetic properties. In a click approach, disulfide re-bridging by a pyridazinedione construct yields a homogeneous porphyrin-PIC with a constant DAR4.

Figure 2

Characterization of photo-immunoconjugates (PICs). (A) The table shows products of 7B2 MAb conjugated with different molar concentrations of IR700, rebridged antibody with pyridazinedione and porphyrin. Chemical characterization by UV-Vis spectrophotometry shows the antibody to PS ratio (DAR). In DLS studies, the zeta potential (ζ) was estimated according to the Huckel equation. The increase in the molecular weights (MWs) of conjugations, measured by microcapillary electrophoresis, are in confirmation of determined DARs. (B) ELISA plates coated with gp41 antigen, as a peptide representing 7B2′s epitope. Results are representative of means of triplicate values with three individual experiments. 924 MAb was used as an isotype control. (C) Microcapillary electrophoresis of reduced and non-reduced 7B2 MAb and PICs, before and after irradiation. Unlike 7B2 and 7B2-IR700, the reduced 7B2-porphyrin did not show the separation of light and heavy chains, confirming the accuracy of click chemistry with DAR4. Size standards are indicated on the side of each “gel”. (D) DLS Histograms of hydrodynamic radius (R_h) for 7B2 MAb and PICs, monitoring how the R_h preserved during the conjugation process. When IR700 was conjugated with an antibody molecule, ligand dissociation from IR700 affected the shape and solubility of the antibody to which it is attached as well as antibody−antigen complexes.

Figure 3

The flow cytometry histogram of PICs by using 293T control cells and Env-transfected 293T cells incubated in PBS + 1% BSA + sodium azide (0.2%). (A) PIC binding to 293T cells transfected with 92UG037.8 gp160 (293T/92UG) cells was directly detected through red fluorescent emission from IR700-PICs and porphyrin-PIC, by filter Qdot 705 and filter Qdot 655, respectively. (B) By using FITC-secondary immunofluorescence, PICs showed equal binding ability into Env-transfected cells, while 7B2-IR700 DAR4 showed somewhat unspecific binding into the control 293T cells. Results are representative of at least three independent experiments. Isotype control (chimeric RAC18) is shown as blue shaded histogram. (C) Studying the binding ability of porphyrin-7B2, in the presence and absence of soluble CD4 (sCD4).

Figure 4

ROS generation and apoptosis assay by PICs. (A) The diagram demonstrates a broad spectrum of light (380−780 nm) by a homemade LED device (30 mW/cm²) for irradiation of both IR700 and porphyrin PICs, at the same time. Blue spectra can excite the Soret band of porphyrin at 432 nm, meanwhile red spectra excite 7B2-IR700 at 689 nm. (B) Samples were kept in the dark (represented by grey) or were irradiated with 50 J/cm². (C) As a positive control, 293T cells were treated with 80 µM H2O2 for 24 h in dark. Control group (without H2O2) was incubated under the same conditions. (D) In apoptosis assay by Annexin V - FITC/PI, the Env-transfected cells were incubated with PICs for 4 h, then irradiated. PICs showed cell death due to late apoptosis (purple color in subset Upper Right) after 4 h incubation. Control 7B2 antibody showed neither cell death nor apoptosis sign. (n = 3).

Figure 5

Comparing the photo-cytotoxicity and targeting of PICs by direct cytotoxicity assay. (A) Env-transfected 293 T cells (293 T/92UG) and control 293T cells were incubated with 7B2-IR700 (DAR 3), 7B2-porphyrin and an equal molarity mixture of both PICs in the presence of sCD4 (anti-gp120) for 4 h then were assayed by PIT. Data are ± means S.E.M. (n = 3) with three individual experiments. Where no error bars are visible, they are obscured by the symbol. (B) An equal molarity mixture of porphyrin-PIC with IR700-PIC may increase the cytotoxicity in compare to porphyrin-PIC or the average cytotoxicity of both PICs. Data are ± means S.E.M. (n = 3). (C) Incubation of transfected cells in two different times (1 and 4 h) before PIT showed the internalization-dependency of Mab-porphyrin to have more photo-cytotoxicity, while the membrane binding of 7B2-IR700 was sufficient to induce cell death. Data are ± means S.E.M. (n = 3).

Figure 6

Target-specific cell death in response to PIT in HIV Env-expressing 293T cells. The cells were treated with IR700-7B2 (A) or 7B2-porphyrin (B) in the presence of sCD4 for 1 h before laser irradiation by microscope. (A) The emitted red fluorescent from IR700-7B2 was directly detected. (B) 7B2-porphyrin was detected indirectly by using FITC anti-human IgG secondary antibody. After 30 min irradiation, the cell viability was studied by using Acridine Orange and Ethidium Bromide (Live/dead dye). White arrows indicate the bleb formation caused the necrotic cell death. The white bar indicates 10 µm. (C) The red square shows the irradiated region that the cells are dead (red color). The non-irradiated region (green color) signifies live cells, confirming no dark toxicity by PIC. The white bar indicates 20 µm.